Membrane transport systems are involved in a number of fundamental cellular processes, and yet most are poorly characterized at the molecular level. The glucose transporter is the prototype facilitative transport protein. Facilitative glucose transport is a critical function carried out by virtually all mammalian cells. This process is mediated by the Glut family of membrane glycoproteins containing six members. The Gluts are responsible for the exchange of glucose between the blood and the cytoplasm of cells, supplying glucose for energy metabolism and the biosynthesis of sugar-containing macromolecules. Additionally, glucose transport in certain tissues plays a critical role in organismal glucose homeostasis. Despite the physiologic importance of glucose transport, relatively little is known concerning the structure and function of the Glut proteins. The long-term goals of this project are to delineate: l) the structure of the facilitative glucose transporter; 2) how that structure is generated during the process of protein biosynthesis; and 3) the molecular mechanism of sugar transport. In the first project period of this grant the 12 transmembrane segment model for the topology of Glut1 was experimentally verified and several residues critical for transport activity were identified and characterized. Additionally, the first dysfunctional mutation in a human Glut gene was discovered in Glut2 of a type II diabetic patient, suggesting a direct involvement of Glut defects in the pathogenesis of diabetes. To make further progress towards accomplishing our long-term goals, the following specific aims are proposed for the next project period: l. Three residues critical for Glut1 transport activity have been identified by site-directed mutagenesis. The function of amino acid residues Q161, V165, and W412 will be explored in detail by amino acid substitution in conjunction with inhibitor and kinetic analyses of the resulting mutant transporters expressed in Xenopus oocytes. 2. Site-directed mutagenesis and affinity-ligand studies implicate transmembrane helices 5, 7, and 11 of Glut1 in the formation of an aqueous chamber for substrate passage through the lipid bilayer. A systematic cysteIne-scanning mutagenesis analysis of the 63 residues within helices 5, 7, and 11 of a cysteine-less Glut1 molecule will be performed to define the role of specific residues within these helices in transport function. This analysis will be combined with the use of cysteine-specific modifying reagents to ascertain the accessibility and relative orientation of residues within the helices. 3. The "charge-difference" hypothesis has been proposed to explain the topology of eucaryotic membrane proteins. However, the hypothesis has not yet been experimentally tested on a native polytopic membrane protein. A glycosylation-scanning mutagenesis procedure has been developed that allows the detailed analysis of Glut1 membrane topology. This procedure will be used in conjunction with site-directed mutagenesis to test the "charge-difference" hypothesis and to explore the structural requirements for proper insertion of Glut1 in the membrane.